Metabolic flux analysis and the NAD(P)H/NAD(P)+ ratios in chemostat cultures of Azotobacter vinelandii

Abstract

BackgroundAzotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P)+ ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions.ResultsThe results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP+ ratio, showing that at the lowest OTR (2.4 mmol L−1 h−1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L−1 h−1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L−1 h−1. At the highest OTR condition, a decrease in the NADPH/NADP+ ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH).ConclusionsWe have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.

Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion

“…In order to obtain quantitative insight into the global carbon fluxes in A. vinelandii , we developed a metabolic model that describes isotope labeling of metabolites upon the addition of 20% universally labeled 13 C-glucose and 80% unlabeled glucose (see Materials and Methods for modeling details). The biochemical reactions and biomass equation adopted in our model come from Andres’s work 16 and are listed in Supplemental Table 1, which is based on genomic information and involves validated pathways that have been identified by our isotope tracer experiments as described above. Specifically, the principal metabolic network includes the ED pathway, the EMP pathway, the OPP pathway, anaplerotic reactions, and the TCA cycle, all of which have been verified by isotope tracer experiments based on this work.…”

“…Primary metabolic network of A. vinelandii was reconstructed according to García et al . 16 and provided in Supplemental Table 1. Functions between free metabolic fluxes and intermediate MDVs were established according to mass balance from reaction stoichiometry and isotopomer balance from atom transition using an elementary metabolic unit (EMU) method 35 .…”

Productivity and scale‐up of poly(3‐hydroxybutyrate) production under different oxygen transfer conditions in cultures of Azotobacter vinelandii

“…With the exception of extended batch cultures at the lowest OTR (6.8 mmol L −1 h −1 ), the results obtained were in line with previous evidence, 6,23 for which an increase in the OTR decreased PHB accumulation. In this sense, García et al 7 . reported that changes in the OTR have an effect on the metabolic fluxes and intracellular NADPH/NADP + ratio.…”

“…With the exception of extended batch cultures at the lowest OTR (6.8 mmol L −1 h −1 ), the results obtained were in line with previous evidence, 6,23 for which an increase in the OTR decreased PHB accumulation. In this sense, García et al 7 reported that changes in the OTR have an effect on the metabolic fluxes and intracellular NADPH/NADP + ratio. Those authors demonstrated that in chemostat cultures at the lowest OTR evaluated (2.4 mmol L −1 h −1 ), the flux through the tricarboxylic acid cycle decreased approximately 28-fold, and the flux through PHB biosynthesis increased almost 7-fold compared to cultures developed at an OTR of 14.6 mmol L −1 h −1 .…”

Quantitative models of nitrogen-fixing organisms

“…The study provided a quantitative baseline for modeling the direct and indirect costs of N 2 fixation more generally. During the similar time period, FBA was applied to Azotobacter and showed that O 2 availability affects TCA cycle, PP pathway and alginate and P3HB (poly-3-hydroxybutyrate) biosynthetic fluxes [164] .…”